Many of the biologically active peptides used as signaling molecules within the endocrine, nervous and immune systems are initially synthesized as larger precursors known as prohormones. These prohormones must undergo post-translational processing to achieve their final, biologically active form. Of the modifications necessary for maturation, the most specific step is the extremely selective cleavage of the prohormone by endoproteases called prohormone converting enzymes (PCEs). In fact, some prohormones are subject to differential processing, the generation of different bioactive peptides from a single precursor, depending on the specific cell types in which they are expressed. This proposal will address two questions: One, is the specificity of the converting enzyme PC2 altered by the presence of neuropeptide 7B2 during its post-translational maturation? Two, can we define the structural elements of a prohormone, which are recognized by specific prohormone converting enzymes? To address this issue, we have successfully developed the necessary technical tools (e.g., purified prohormones and cleavage intermediates, purified converting enzymes, and an in vitro assay capable of producing defined products) to examine the processing of both proinsulin and proglucagon by the converting enzymes PC1 and PC2. Using these tools, we will examine the specificity of PC2, synthesized in the presence or absence of neuropeptide 7B2, using both proglucagon and proinsulin as substrates. Following this, we will examine the ability of both PC1 and PC2 to recognize and cleave a series of altered forms of proinsulin. Using the results of these studies as a guide, we will then attempt to transfer elements of proinsulin cleavage site specificity to proglucagon and its conversion intermediates. Results from both the currently proposed and future studies should provide us with significant insights into the mechanisms of enzyme-substrate specificity and protein-protein interactions. This knowledge will hopefully prove useful not only in understanding prohormone processing and the broader area of islet cell physiology, but in other areas where proteases play a significant role in both normal homeostasis and the pathologic basis of disease.